Investigating SARS-CoV-2 breakthrough infections per variant and vaccine type.

Breakthrough SARS-CoV-2 infections have been reported in fully vaccinated individuals, in spite of the high efficacy of the currently available vaccines, proven in trials and real-world studies. Several variants of concern (VOC) have been proffered to be associated with breakthrough infections following immunization. In this study, we investigated 378 breakthrough infections recorded between January and July 2021 and compared the distribution of SARS-CoV-2 genotypes identified in 225 fully vaccinated individuals to the frequency of circulating community lineages in the region of South Limburg (The Netherlands) in a week-by-week comparison. Although the proportion of breakthrough infections was relatively low and stable when the Alpha variant was predominant, the rapid emergence of the Delta variant lead to a strong increase in breakthrough infections, with a higher relative proportion of individuals vaccinated with Vaxzevria or Jcovden being infected compared to those immunized with mRNA-based vaccines. A significant difference in median age was observed when comparing fully vaccinated individuals with severe symptoms (83 years) to asymptomatic cases (46.5 years) or individuals with mild-to-moderate symptoms (42 years). There was no association between SARS-CoV-2 genotype or vaccine type and disease symptoms. Furthermore, the majority of adaptive mutations were concentrated in the N-terminal domain of the Spike protein, highlighting its role in immune evasion. Interestingly, symptomatic individuals harbored significantly higher SARS-CoV-2 loads than asymptomatic vaccinated individuals and breakthrough infections caused by the Delta variant were associated with increased viral loads compared to those caused by the Alpha variant. In addition, we investigated the role of the Omicron variant in causing breakthrough infections by analyzing 135 samples that were randomly selected for genomic surveillance during the transition period from Delta to Omicron. We found that the proportion of Omicron vs. Delta infections was significantly higher in individuals who received a booster vaccine compared to both unvaccinated and fully vaccinated individuals. Altogether, these results indicate that the emergence of the Delta variant and in particular Omicron has lowered the efficiency of particular vaccine types to prevent SARS-CoV-2 infections and that, although rare, the elderly are particularly at risk of becoming severely infected as the consequence of a breakthrough infection.

Culture-Independent Genotyping Improves Surveillance of Neisseria gonorrhoeae, Especially in Oropharyngeal Samples, the Netherlands, 2017 to 2018.

It is important i to monitor the transmission and antimicrobial resistance of Neisseria gonorrhoeae (NG). Current surveillance relies on culturing, which frequently fails. Previously, a culture-independent genotyping method was developed based on NG multi-antigen sequence typing (NG-MAST). To determine whether crucial sequence types (STs) are missed during culture-dependent surveillance, NG-positive NAAT samples were genotyped, and the results of the culture-positive and culture-negative samples were compared. In total, 196 NG-positive NAAT samples, from January 2017 until August 2018, which were also routinely cultured, were retrospectively included. Genotyping was successful in 152 NAAT samples (77.0%), 33 NAAT samples failed, and 11 NAAT samples showed possible mixed strain infections. Oropharyngeal samples (n = 16) showed the largest increase in typing rate from 6.3% (1/16) success in culture-dependent genotyping to 81.3% (13/16) in culture-independent genotyping. Nine genogroups (n ≥ 5 samples) were found; all included both culture-positive and culture-negative NG. However, culture-independent surveillance revealed 14 additional STs in the culture-negative samples. Overall, culture-dependent surveillance could detect all genogroups, indicating that major trends could be identified with culture-dependent surveillance. However, culture-independent surveillance provides more STs, mixed infections and more oropharyngeal samples, giving a more detailed view and could result in an earlier detection of outbreaks and transmission.

Increased transmissibility of SARS-CoV-2 alpha variant (B.1.1.7) in children: three large primary school outbreaks revealed by whole genome sequencing in the Netherlands.

BACKGROUND: Variant of concern (VOC) SARS-CoV-2 alpha variant (B.1.1.7) was the dominant strain in the Netherlands between March 2021-June 2021. We describe three primary school outbreaks due to the alpha variant using whole genome sequencing with evidence of large-scale transmission among children, teachers and their household contacts. METHOD: All outbreaks described were investigated by the South Limburg Public Health Service, the Netherlands. A case was defined as an individual with a real-time polymerase chain reaction test or antigen test positive for SARS-CoV-2. Whole genome sequencing was performed on random samples from at least one child and one teacher of each affected class. RESULTS: Peak attack rates in classes were 53%, 33% and 39%, respectively. Specific genotypes were identified for each school across a majority of affected classes. Attack rates were high among staff members, likely to promote staff-to-children transmission. Cases in some classes were limited to children, indicating child-to-child transmission. At 39%, the secondary attack rate (SAR) in household contacts of infected children was remarkably high, similar to SAR in household contacts of staff members (42%). SAR of household contacts of asymptomatic children was only 9%. CONCLUSION: Our findings suggest increased transmissibility of the alpha variant in children compared to preceding non-VOC variants, consistent with a substantial rise in the incidence of cases observed in primary schools and children aged 5-12 since the alpha variant became dominant in March 2021. Lack of mandatory masking, insufficient ventilation and lack of physical distancing also probably contributed to the school outbreaks. The rise of the delta variant (B.1.617.2) since July 2021 which is estimated to be 55% more transmissible than the alpha variant, provides additional urgency to adequate infection prevention in school settings.

Infections with the SARS-CoV-2 Delta variant exhibit fourfold increased viral loads in the upper airways compared to Alpha or non-variants of concern.

There has been a growing body of evidence that the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Delta variant (B.1.617.2) shows enhanced transmissibility and increased viral loads compared to other variants. A recent study has even suggested that respiratory samples from people infected with the Delta variant can harbor up to 1000 times higher viral loads compared to samples with variants that are more closely related to the original Wuhan strain, although the sample size of this study (n = 125) was very limited. Here, we have compared the viral load in 16,185 samples that were obtained in periods during which non-VOC, the Alpha (B.1.1.7) or Delta variant (B.1.617.2) were dominant as evidenced by genomic surveillance. We found that the Delta variant contained about fourfold higher viral loads across all age groups compared to the non-VOC or Alpha variants, which is significantly lower than reported earlier. Interestingly, the increased viral load for the Delta variant seemed to be age-dependent, regardless of sex, as the viral load was about 14-fold higher for Delta compared to the non-VOC or Alpha variant in age group 0-20 years and fourfold higher in age group 21-40 years, while there was no difference in viral load between variants in age groups 41-60 and 61+ years, most likely as a consequence of a higher degree of vaccination in the older age groups.

Linked nosocomial COVID-19 outbreak in three facilities for people with intellectual and developmental disabilities due to SARS-CoV-2 variant B.1.1.519 with spike mutation T478K in the Netherlands.

BACKGROUND: Individuals with intellectual and developmental disabilities (IDD) living in congregated settings have increased risk of COVID-19 infection and mortality. Little is known about variant B.1.1.519 with spike mutation T478K, dominant in Mexico. We describe a linked SARS-CoV-2 B.1.1.519 outbreak in three IDD facilities in the Netherlands. METHODS: Following notification of the index, subsequent cases were identified through serial PCR group testing. Positive specimens were submitted for whole-genome-sequencing. Clinical information was gathered through interviews with staff members of the three facilities. RESULTS: Attack rate (AR) in clients of the index facility was 92% (23/25), total AR in clients 45% (33/73) and in staff members 24% (8/34). 55% (18/33) of client cases were asymptomatic, versus 25% (2/8) of staff members. Five client cases (15%) were hospitalized, two died (6%). Sequencing yielded the same specific B.1.1.519 genotype in all three facilities. No significant difference in median viral load was established comparing the B.1.1.519 variant with other circulating variants. The index of the linked outbreak reported no travel history or link to suspected or confirmed cases suggesting regional surveillance. Observed peak regional prevalence of B.1.1.519 during the outbreak supports this. CONCLUSION: AR, morbidity and mortality prior to control measures taking effect were high, probably related to the specific characteristics of the IDD setting and its clients. We assessed no evidence for intrinsic contributing properties of variant B.1.1.519. Our study argues for enhanced infection prevention protocols in the IDD setting, and prioritization of this group for vaccination against COVID-19.

Genetic analysis of plasmid-encoded mcr-1 resistance in Enterobacteriaceae derived from poultry meat in the Netherlands.

BACKGROUND: Colistin is classified as the highest priority and critically important antimicrobial for human medicine by WHO as it is the last resort agent for treatment of carbapenem-resistant Enterobacteriaceae in humans. Additional research is necessary to elucidate the genetic structure of mcr-1 resistance genes, commonly found on plasmids, using WGS. OBJECTIVES: To map and compare the genetic characteristics of 35 mcr-1-mediated colistin-resistant Enterobacteriaceae isolated from chicken meat to highlight the genetic variation of the mcr-1-containing plasmids. METHODS: Sequencing was performed using Illumina HiSeq2500, Novaseq6000 and ONT's GridION. GridION data was locally basecalled and demultiplexed using ONT's Albacore 2.3.4 followed by Porechop 2.3. Quality filtering was performed using Filtlong 2.0. Hybrid Assembly was performed using Unicycler 4.7. Plasmids were compared with reference sequences in plasmid-RefSeq and pATLAS. RESULTS: A total of 35 mcr-1 positive Enterobacteriaceae were investigated, which resulted in 34 qualitatively robust hybrid assemblies of 2 Klebsiella pneumoniae and 32 Escherichia coli. mcr-1.1 was present in 33/34 isolates. One isolate contained an mcr-1.1-like resistance gene, due to a deletion of one codon. Two mcr-1.1 genes were located on the chromosome, while the majority of the mcr-1 genes were found on IncX4 type plasmids (n = 19). Almost all plasmids identified in this study were highly similar to plasmids found in human-derived strains. CONCLUSIONS: The mcr-1.1-containing plasmids from retail chicken show high sequence similarity to human mcr-1.1 plasmids, suggesting that this may be a contributor to the presence of colistin resistance in humans.

Centre-specific bacterial pathogen typing affects infection-control decision making.

Whole-genome sequencing is becoming the de facto standard for bacterial outbreak surveillance and infection prevention. This is accompanied by a variety of bioinformatic tools and needs bioinformatics expertise for implementation. However, little is known about the concordance of reported outbreaks when using different bioinformatic workflows. In this multi-centre proficiency testing among 13 major Dutch healthcare-affiliated centres, bacterial whole-genome outbreak analysis was assessed. Centres who participated obtained two randomized bacterial datasets of Illumina sequences, a Klebsiella pneumoniae and a Vancomycin-resistant Enterococcus faecium, and were asked to apply their bioinformatic workflows. Centres reported back on antimicrobial resistance, multi-locus sequence typing (MLST), and outbreak clusters. The reported clusters were analysed using a method to compare landscapes of phylogenetic trees and calculating Kendall-Colijn distances. Furthermore, fasta files were analysed by state-of-the-art single nucleotide polymorphism (SNP) analysis to mitigate the differences introduced by each centre and determine standardized SNP cut-offs. Thirteen centres participated in this study. The reported outbreak clusters revealed discrepancies between centres, even when almost identical bioinformatic workflows were used. Due to stringent filtering, some centres failed to detect extended-spectrum beta-lactamase genes and MLST loci. Applying a standardized method to determine outbreak clusters on the reported de novo assemblies, did not result in uniformity of outbreak-cluster composition among centres.

Culture-Independent Genotyping Revealed 3 Strain Clusters in a Potential Neisseria gonorrhoeae Outbreak in Young Heterosexuals (<25 Years), the Netherlands, October 2017 to March 2019.

BACKGROUND: Investigation was undertaken to determine the genetic relatedness of Neisseria gonorrhoeae (NG) isolates of young (<25 years) heterosexuals of a potential outbreak from October 2017 to March 2019 in South-Limburg, the Netherlands. METHODS: Data and residual sample material of routine diagnostics were retrieved for outbreak cases (78/81), young heterosexuals at baseline (January 2016 to September 2017, n = 30), and men who have sex with men (2018, n = 47). Total DNA was isolated, and NG was genotyped using culture-free NG multiantigen sequence typing. Sanger sequence data were used to construct a phylogenetic tree. Cases of outbreak clusters were geographically mapped, and descriptive analyses were performed on patient characteristics, comparing these clusters. RESULTS: Outbreak investigation showed 81 cases of young heterosexuals between October 2017 and March 2019 (4.5 per month) compared with 30 between January 2016 and September 2017 (1.4 per month), which was considered as baseline. Culture-independent genotyping of NG was performed to assess the genetic relatedness, as only 21 outbreak cases were culture confirmed. This revealed 3 independent outbreak clusters G2 (n = 18), G13113 (n = 11), and GNewST (n = 24). None of the clusters were geographically linked or introduced by bridging with men who have sex with men networks. Number of sex partners reported by men and Chlamydia trachomatis coinfection were associated with clusters G2 and GNewST, respectively. CONCLUSIONS: Culture-independent typing proved to be essential to identify the 3 outbreak clusters. However, targeted interventions were difficult because information on sex partners was limited. Therefore, prospective culture-independent typing could be used for early outbreak detection and aid in transmission prevention.

Molecular epidemiological analysis of Mycoplasma genitalium shows low prevalence of azithromycin resistance and a well-established epidemic in South Africa.

OBJECTIVES: Macrolide resistance in Mycoplasma genitalium is emerging globally. There is paucity of data from sub-Saharan Africa where syndromic management is used to treat sexually transmitted infections (STIs). We conducted a molecular epidemiological study to determine the prevalence of azithromycin resistance and epidemic diversity of M. genitalium infections in South Africa. METHODS: We analysed 90 M. genitalium-positive specimens that had been collected consecutively from men and women (50% symptomatic) from geographically diverse communities across the northern part of South Africa between 2015 and 2019. Melting curve analysis followed by targeted sequencing of the 23S rRNA gene was performed to detect azithromycin resistance. Molecular typing was done through single nucleotide polymorphism (SNP) analysis of the MG191 gene and short tandem repeats (STR) assessment of the MG309 gene. An overview of all published M. genitalium sequence types was generated and novel sequence types identified in this study were allocated numbers accordingly. RESULTS: Azithromycin resistance was detected in 1/90 M. genitalium-positive specimens (1.1%; 95% CI 0% to 3.3%) as conferred by A2071G mutation; this strain also harboured a C234T mutation in the parC gene with wild type gyrA gene. SNP typing and STR assessment was successful in 38/90 specimens (42%) and showed a genetically diverse epidemic, without geographic clustering, with eight novel sequence types identified. CONCLUSION: This is the first study that determines resistance in M. genitalium infection since introduction of azithromycin in the syndromic management regimen for STIs in South Africa in 2015. Despite a well-established epidemic, azithromycin-resistant M. genitalium infection is still uncommon in the public healthcare sector. However, it has the potential to undermine the effectiveness of syndromic management. Introduction of molecular diagnostics and continuous surveillance are warranted for early detection emergence of resistance.

Antimicrobial Resistance of Neisseria gonorrhoeae Isolates from High-Risk Men in Johannesburg, South Africa.

Neisseria gonorrhoeae antimicrobial drug resistance has emerged worldwide; however, the situation in sub-Saharan Africa is not well documented. We investigated the molecular epidemiology and occurrence of antimicrobial resistance in Neisseria gonorrhoeae infections in two core transmission groups of men in Johannesburg, South Africa. We recruited men who have sex with men (MSM) presenting with urethral discharge and men with recurrent episodes of urethral discharge. Molecular testing and culture for N. gonorrhoeae were performed, followed by antimicrobial susceptibility testing. Whole-genome sequencing (WGS) was used to identify resistance-conferring mutations and to determine the genetic relatedness of the isolates. In all, 51 men were recruited; 42 (82%) had N. gonorrhoeae infections. Most gonococcal isolates were resistant to ciprofloxacin (78%) and tetracycline (74%); 33% were penicillin resistant. All gonococcal isolates were susceptible to cephalosporins and spectinomycin. Azithromycin resistance was observed in 4 (15%) isolates (epidemiological cutoff), all with mutations in the mtrR promoter region. Most of the isolates (19/27) harbored the gonococcal genetic island, which is associated with antimicrobial resistance. WGS revealed a diverse epidemic with mostly novel NG-STAR (70%) and NG-MAST (70%) sequence types. Thus, we demonstrate a high prevalence of antimicrobial resistance in Neisseria gonorrhoeae strains obtained from high-risk men in South Africa. The introduction of diagnostics and scale-up of surveillance are warranted to prevent the emergence of multidrug-resistant infections.

Men and Women Have Similar Neisseria gonorrhoeae Bacterial Loads: a Comparison of Three Anatomical Sites.

Neisseria gonorrhoeae is a common bacterial sexually transmitted infection (STI). Currently, there are limited data on the bacterial load in both men and women and on both genital and extragenital sites. Therefore, we quantified N. gonorrhoeae bacterial loads in a large population of women, heterosexual men, and men who have sex with men (MSM) at three different anatomical sites. N. gonorrhoeae-positive samples (n = 1265) of STI clinic consultations (n = 944) were tested for N. gonorrhoeae with the Roche Cobas 4800 system, and quantification cycle (Cq) values were used as an inversely proportional measure for N. gonorrhoeae bacterial load after interpolation from a standard curve. Bacterial loads were compared between sample materials and sexes using t tests. The following mean N. gonorrhoeae loads were observed: urine, 4.5 ± 1.0 log10 CFU/ml; vaginal swabs, 4.3 ± 1.1 log10 CFU/ml; anorectal swabs (women), 4.0 ± 1.2 log10 CFU/ml; anorectal swabs (men), 4.5 ± 1.3 log10 CFU/ml; oropharyngeal swabs (women), 2.8 ± 0.9 log10 CFU/ml; and oropharyngeal swabs (men), 3.2 ± 1.0 log10 CFU/ml. Oropharyngeal swabs had a significantly lower N. gonorrhoeae load (P < 0.001) than genital and anorectal samples. Loads did not differ between men and women. This is the first study that determined N. gonorrhoeae load in both women and men at three anatomical sites. The substantial N. gonorrhoeae load at all sample sites suggest that all sites may have transmission potential. However, the oropharyngeal site presents the lowest bacterial load. Men and women have a similar N. gonorrhoeae loads on separate anatomical sites, arguing for similar transmission potential and similar clinical relevance.

CT in relation to RT-PCR in diagnosing COVID-19 in The Netherlands: A prospective study.

INTRODUCTION: Early differentiation between emergency department (ED) patients with and without corona virus disease (COVID-19) is very important. Chest CT scan may be helpful in early diagnosing of COVID-19. We investigated the diagnostic accuracy of CT using RT-PCR for SARS-CoV-2 as reference standard and investigated reasons for discordant results between the two tests. METHODS: In this prospective single centre study in the Netherlands, all adult symptomatic ED patients had both a CT scan and a RT-PCR upon arrival at the ED. CT results were compared with PCR test(s). Diagnostic accuracy was calculated. Discordant results were investigated using discharge diagnoses. RESULTS: Between March 13th and March 24th 2020, 193 symptomatic ED patients were included. In total, 43.0% of patients had a positive PCR and 56.5% a positive CT, resulting in a sensitivity of 89.2%, specificity 68.2%, likelihood ratio (LR)+ 2.81 and LR- 0.16. Sensitivity was higher in patients with high risk pneumonia (CURB-65 score ≥3; n = 17, 100%) and with sepsis (SOFA score ≥2; n = 137, 95.5%). Of the 35 patients (31.8%) with a suspicious CT and a negative RT-PCR, 9 had another respiratory viral pathogen, and in 7 patients, COVID-19 was considered likely. One of nine patients with a non-suspicious CT and a positive PCR had developed symptoms within 48 hours before scanning. DISCUSSION: The accuracy of chest CT in symptomatic ED patients is high, but used as a single diagnostic test, CT can not safely diagnose or exclude COVID-19. However, CT can be used as a quick tool to categorize patients into "probably positive" and "probably negative" cohorts.

Appearance of vanD-positive Enterococcus faecium in a tertiary hospital in the Netherlands: prevalence of vanC and vanD in hospitalized patients.

Vancomycin-resistant enterococci (VRE) can rapidly spread through hospitals. Therefore, our hospital employs a screening program whereby rectal swabs are screened for the presence of vanA and vanB, and only PCR-positive broths are cultured on VRE selection agar. Early November 2016, a clinical vanA-/vanB-negative VRE isolate was detected in a vanA/vanB-screening-negative patient, giving the possibility that an undetected VRE might be spreading within our hospital. Whole-genome-sequencing of the isolate showed that resistance was vanD-mediated and core genome multilocus sequence typing showed it was a rare type: ST17/CT154. To determine the prevalence of vanA/B/C/D-carrying enterococci, we designed a real-time PCR for vanC1/2/3 and vanD and screened rectal swabs from 360 patients. vanD was found in 27.8% of the patients, yet culture demonstrated only E. faecium from vanA-positive broths and E. gallinarum from vanC1-positive broths. No vanD-positive VRE were found, limiting the possibility of nosocomial spread of this VRE. Moreover, the high prevalence of non-VRE vanD in rectal swabs makes it unfeasible to include the vanD PCR in our VRE screening. However, having validated the vanC1/2/3 and vanD PCRs allows us to rapidly check future vanA/B-negative VRE for the presence of vanC and vanD genes.

Culture-free genotyping of Neisseria gonorrhoeae revealed distinct strains at different anatomical sites in a quarter of patients, the Netherlands, 2012 to 2016.

BackgroundGenotyping of Neisseria gonorrhoeae (NG) is essential for surveillance to monitor NG transmission and dissemination of resistant strains. Current genotyping methods rely on bacterial culture which frequently fails.AimOur aim was to develop a culture-free genotyping method that is compatible with the widely used N. gonorrhoeae multi-antigen sequence typing (NG-MAST) database, which facilitates genotyping of NG detected at separate anatomical sites in individual patients.MethodsSpecific primers for both PCR targets porB and tbpB were designed and technically validated by assessing the analytical sensitivity, cross-reactivity with 32 non-gonoccocal Neisseria species, and concordance with NG-MAST. Clinical application was assessed on 205 paired samples from concurrent NG infections at different anatomical sites of 98 patients (81 men who have sex with men and 17 women) visiting our sexually transmitted infections clinic.ResultsTyping could be consistently performed on samples with a PCR quantification cycle (Cq) value <35. Furthermore, the method showed no cross-reactivity and was concordant with NG-MAST. Culture-free NG-MAST improved the typing rate from 62% (59/95) for cultured samples to 94% (89/95) compared with culture-dependent NG-MAST. Paired samples of 80 of 98 patients were genotyped, revealing distinct NG strains in separate anatomical sites in 25% (20/80) of the patients.ConclusionsThis NG-specific genotyping method can improve NG surveillance as it facilitates genotyping of non-culturable and extra-genital samples. Furthermore, 25% of patients were infected with multiple NG strains, which is missed in current culture-dependent surveillance. Including non-culturable and concurrent NG infections in surveillance informs actions on dissemination of multidrug-resistant NG strains.

Detection of the plasmid-mediated colistin-resistance gene mcr-1 in faecal metagenomes of Dutch travellers.

BACKGROUND: Recently, the first plasmid-mediated colistin-resistance gene, mcr-1, was reported. Colistin is increasingly used as an antibiotic of last resort for the treatment of infections caused by carbapenem-resistant bacteria, which have been rapidly disseminating worldwide in recent years. OBJECTIVES: The reported carriage rate of mcr-1 in humans remains sporadic thus far, except for those reported in Chinese populations. We aimed to determine its presence in the faecal metagenomes of healthy Dutch travellers between 2010 and 2012. METHODS: Faecal metagenomic DNA of pre- and post-travel samples from 122 healthy Dutch long-distance travellers was screened for the presence of mcr-1 using a TaqMan quantitative PCR assay, which was designed in this study. All positive samples were confirmed by sequencing of the amplicons. RESULTS: The mcr-1 gene was detected in 6 (4.9%, 95% CI = 2.1%-10.5%) of 122 healthy Dutch long-distance travellers after they had visited destinations in South(-east) Asia or southern Africa between 2011 and 2012. One of these participants was already found to be positive before travel. CONCLUSIONS: Our study highlights the potential of PCR-based targeted metagenomics as an unbiased and sensitive method to screen for the carriage of the mcr-1 gene and suggests that mcr-1 is widespread in various parts of the world. The observation that one participant was found to be positive before travel suggests that mcr-1 may already have disseminated to the microbiomes of Dutch residents at a low prevalence, warranting a more extensive investigation of its prevalence in the general population and possible sources.

Dissemination of Antimicrobial Resistance in Microbial Ecosystems through Horizontal Gene Transfer.

The emergence and spread of antibiotic resistance among pathogenic bacteria has been a rising problem for public health in recent decades. It is becoming increasingly recognized that not only antibiotic resistance genes (ARGs) encountered in clinical pathogens are of relevance, but rather, all pathogenic, commensal as well as environmental bacteria-and also mobile genetic elements and bacteriophages-form a reservoir of ARGs (the resistome) from which pathogenic bacteria can acquire resistance via horizontal gene transfer (HGT). HGT has caused antibiotic resistance to spread from commensal and environmental species to pathogenic ones, as has been shown for some clinically important ARGs. Of the three canonical mechanisms of HGT, conjugation is thought to have the greatest influence on the dissemination of ARGs. While transformation and transduction are deemed less important, recent discoveries suggest their role may be larger than previously thought. Understanding the extent of the resistome and how its mobilization to pathogenic bacteria takes place is essential for efforts to control the dissemination of these genes. Here, we will discuss the concept of the resistome, provide examples of HGT of clinically relevant ARGs and present an overview of the current knowledge of the contributions the various HGT mechanisms make to the spread of antibiotic resistance.

A Flagellar Glycan-Specific Protein Encoded by Campylobacter Phages Inhibits Host Cell Growth.

We previously characterized a carbohydrate binding protein, Gp047, derived from lytic Campylobacter phage NCTC 12673, as a promising diagnostic tool for the identification of Campylobacter jejuni and Campylobacter coli. We also demonstrated that this protein binds specifically to acetamidino-modified pseudaminic acid residues on host flagella, but the role of this protein in the phage lifecycle remains unknown. Here, we report that Gp047 is capable of inhibiting C. jejuni growth both on solid and liquid media, an activity, which we found to be bacteriostatic. The Gp047 domain responsible for bacterial growth inhibition is localized to the C-terminal quarter of the protein, and this activity is both contact- and dose-dependent. Gp047 gene homologues are present in all Campylobacter phages sequenced to date, and the resulting protein is not part of the phage particle. Therefore, these results suggest that either phages of this pathogen have evolved an effector protein capable of host-specific growth inhibition, or that Campylobacter cells have developed a mechanism of regulating their growth upon sensing an impending phage threat.

A receptor-binding protein of Campylobacter jejuni bacteriophage NCTC 12673 recognizes flagellin glycosylated with acetamidino-modified pseudaminic acid.

Bacteriophage receptor-binding proteins (RBPs) confer host specificity. We previously identified a putative RBP (Gp047) from the campylobacter lytic phage NCTC 12673 and demonstrated that Gp047 has a broader host range than its parent phage. While NCTC 12673 recognizes the capsular polysaccharide (CPS) of a limited number of Campylobacter jejuni isolates, Gp047 binds to a majority of C. jejuni and related Campylobacter coli strains. In this study, we demonstrate that Gp047 also binds to acapsular mutants, suggesting that unlike the parent phage, CPS is not the receptor for Gp047. Affinity chromatography and far-western analyses of C. jejuni lysates using Gp047 followed by mass spectrometry indicated that Gp047 binds to the major flagellin protein, FlaA. Because C. jejuni flagellin is extensively glycosylated, we investigated this binding specificity further and demonstrate that Gp047 only recognizes flagellin decorated with acetamidino-modified pseudaminic acid. This binding activity is localized to the C-terminal quarter of the protein and both wild-type and coccoid forms of C. jejuni are recognized. In addition, Gp047 treatment agglutinates vegetative cells and reduces their motility. Because Gp047 is highly conserved among all campylobacter phages sequenced to date, it is likely that this protein plays an important role in the phage life cycle.

The application of new molecular methods in the investigation of a waterborne outbreak of norovirus in Denmark, 2012.

In December 2012, an outbreak of acute gastrointestinal illness occurred in a geographical distinct area in Denmark covering 368 households. A combined microbiological, epidemiological and environmental investigation was initiated to understand the outbreak magnitude, pathogen(s) and vehicle in order to control the outbreak. Norovirus GII.4 New Orleans 2009 variant was detected in 15 of 17 individual stool samples from 14 households. Norovirus genomic material from water samples was detected and quantified and sequencing of longer parts of the viral capsid region (>1000 nt) were applied to patient and water samples. All five purposely selected water samples tested positive for norovirus GII in levels up to 1.8×10(4) genomic units per 200 ml. Identical norovirus sequences were found in all 5 sequenced stool samples and 1 sequenced water sample, a second sequenced water sample showed 1 nt (<0.1%) difference. In a cohort study, including 256 participants, cases were defined as residents of the area experiencing diarrhoea or vomiting onset on 12-14 December 2012. We found an attack rate of 51%. Being a case was associated with drinking tap-water on 12-13 December (relative risk = 6.0, 95%CI: 1.6-22) and a dose-response relation for the mean glasses of tap-water consumed was observed. Environmental investigations suggested contamination from a sewage pipe to the drinking water due to fall in pressure during water supply system renovations. The combined microbiological, epidemiological and environmental investigations strongly indicates the outbreak was caused by norovirus contamination of the water supply system.